A method of quantitative chemiluminescence immunoassay for the concentration of Growth differentiation factor-15

Growth differentiation factor-15 (GDF-15), a member of the transforming growth factor (TGF-β) superfamily, and is expressed and secreted in response to inflammation, oxidative stress and hypoxia. It has been shown in several studies to be a predictor of heart failure. However, the only kits available on the market are ELISA kits, which are costly and error-prone and are not conducive for clinical use. Here, we developed a chemiluminescence kit which optimized the reaction conditions and the reaction time was reduced to 10 min. We further proved that it can be used to measure GDF-15 in serum or plasma accurately and fastly, and provide additional information for the diagnosis of heart failure disease. Methodological comparison and clinical study verified this method is a reliable, economical and highly automated blood test method.• All necessary steps and the reagents needed are provided.• Reliability of the chemiluminescence immunoassay was verified by analyzing serum GDF-15 levels from different groups.• GDF-15 can provide clinicians with reliable prediction and disease assessment of heart failure.

• All necessary steps and the reagents needed are provided.
• Reliability of the chemiluminescence immunoassay was verified by analyzing serum GDF-15 levels from different groups.• GDF-15 can provide clinicians with reliable prediction and disease assessment of heart failure.

Subject area:
Immunology and Microbiology More specific subject area: Clinical laboratory diagnostics Name of your method: Chemiluminescence Immunoassay Name and reference of original method: ELISA / J. Yin

Introduction
Due to the population aging, the prevalence and fatality rates of cardiovascular diseases are on the rise.Heart failure, which is the primary cause of death related to cardiovascular diseases, is not an isolated ailment but rather a shared pathway for various heart conditions.Currently, NT-proBNP is the widely employed prognostic biomarker for heart failure (HF).However, NT-proBNP does not encompass the complete pathophysiology of HF, and there may be other biomarkers that can reflect different disease mechanisms [1] .
GDF-15, a member of the transforming growth factor (TGF-) superfamily, is typically expressed in low concentrations across various organs [2] .However, in diseases affecting the heart [3] , kidney [4] , lung [5] , and liver [6] , its expression is elevated.As a result, GDF-15 can serve as a potential predictor for systemic processes in heart failure.Currently, only ELISA kits for scientific research are available in the market [7] .These kits are complex, prone to errors, and exhibit low sensitivity.To address these limitations, we have developed a chemiluminescence kit based on streptavidin superparamagnetic particles.This kit offers a larger specific surface area, allowing for more antibody coating.It is also more sensitive and can be easily automated, thereby compensating for the drawbacks of ELISA kits ( Fig. 1 ).The capture antibody was dialyzed using PBS and diluted with PBS to 1mg/ml, 1 mg (1 mL) of the capture antibody after dialysis was taken into the sample tube, and added 3.3 μL 10 mM NHS-LC-LC-Biotin.Incubate the mixture for 30 min at room temperature with stirring.b) After the reaction was completed, 10 μl Tris buffer (1 mol/L) was added, mixed and reacted at room temperature for 10 min.c) Remove the non-reacted and hydrolyzed biotin reagent by desalting Columns.

Materials
(2) Magnetic particle coupling a) Add 2 mL of streptavidin magnetic particles to a normal 15 mL Flacon tube.Make sure all magnetic particles are added to the tube.b) Add 8 mL storage buffer 1 to vial and mix, place it on the magnetic separator, wait for the liquid to clear, discard the supernatant, repeat the above cleaning step three times, then resuspend with 8 ml storage buffer 1. c) Add 40 μL biotinylated capture antibody, mix well, and react on a rocker for 0.5 h at room temperature.d) Repeat buffer wash in step 2(b).e) Add 200 μL blocking reagent to vial and incubate at room temperature on a rocker for 15 min.f) Repeat buffer wash in step 2(b).g) Add 42 mL storage buffer 1 to dilute the magnetic particles concentration to 0.4 mg/ml.
(3) Acridine ester labeling reaction a) The tracer antibody was dialyzed using PBS and diluted with PBS to 1mg/ml, 1 mg (1 mL) of the tracer antibody after dialysis was taken into the sample tube, add 10 μL NSP-DMAE-NHS (10 mM) to sample tube.React and protected from light on a rocker for 1 h at room temperature.b) Add 20 μL l-lysine (10 mg/mL), and mix immediately, and react at room temperature on a blood mixing apparatus for 10 min under the condition of avoiding light.c) The finished tracer antibodies need to be purified using a desalting column to remove excess acridine ester according to manufacturer's instructions.d) Acridine ester labeled tracer antibodies were diluted to 0.1 μg/mL using Storage buffer 2. (4) Reaction process a) The i 3000 Automatic Chemiluminescence Analyzer from Maccura Biotechnology Co., Ltd was used for detection.10 μl sample, 50 μl capture reagent, and 100 μl tracer reagent were added to reaction vials and mixed, After 10 min reacting at 37 • C, 200 μL pre-excitation fluid (H 2 O 2 ) and 200 μL excitation fluid (NaOH) were then added and measured the relative light unit (RLU).Because acridine ester luminescence belongs to the flash type, so our light signal acquisition time is only 100 ms.The concentration of GDF-15 in the sample is proportional to RLU.

Method validation
To analyze the effectiveness of this chemiluminescence immunoassay, we performed methodological comparisons with commercially available ELISA kits (Human GDF-15 Quantikine QuicKit ELISA, Catalog: QK957, R&D systems).A total of 254 samples were used including children, the elderly, pregnant women, dialysis patients and heart patients.We also analyzed serum GDF-15 levels in apparently healthy controls, patients with metabolic syndrome, acute coronary syndrome, atrial fibrillation and chronic heart failure.Mann Whitney test was used for comparison between groups.Spearman's rank correlation was used for correlation analysis.
Fig. 2 shows a comparison of our established chemiluminescence immunoassay with a commercially ELISA kit.Spearman's rank correlation was 0.953 (95 %CI: 0.941-0.963),indicating a significant correlation between the two assay ( P < 0.001).Although the median values of CLIA and ELISA were significantly different, and the GDF-15 concentration of CLIA was significantly higher than that of ELISA.There were significant differences in GDF-15 concentration among different populations.GDF-15 levels were significantly elevated in patients with metabolic syndrome, acute coronary syndrome, atrial fibrillation and chronic heart failure ( Fig. 3 ).These data are consistent with previous findings that GDF-15 levels raise significantly in cardiovascular disease compared to healthy controls [ 1 , 8-10 ].Therefore, we conclude that our chemiluminescence immunoassay is a reliable method for analyzing serum GDF-15 concentration in clinical and in vivo studies.

Ethics statements
The study has been granted an exemption from requiring ethics approval by the Institutional Review Boards and ethics committee at University of science and technology of China as well as the First Affiliated Hospital of USTC, and written informed consent was not required for these reasons: 1.This research using medical records and biological specimens obtained in previous clinical care 2. The risk to the subject of the study is not greater than the minimal risk.

Reagents•Fig. 1 .
Fig. 1.The reaction principle of quantitative chemiluminescence immunoassay for concentration determination of GDF-15. A. Mechanism illustration of the biotin labeling to capture antibody.The N -Hydroxysuccinimide on NHS-LC-LC-Biotin reacts with the primary amino group of the capture antibody to form an amide bond.B. The capture antibody is coupled to the magnetic particle by a specific reaction of biotin and streptavidin.C. The principle of NSP-DMAE-NHS coupling to tracer antibody.D. Detection principle of the chemiluminescence immunoassay.The antigen concentration is proportional to the relative light unit (RLU).

Fig. 2 .
Fig. 2. A, Correlation between GDF-15 levels in 254 serum samples were measured by CLIA vs. ELISA.Correlation coefficient and P-value were calculated using the Spearman's rank correlation.B, Differences in GDF-15 concentrations between CLIA and ELISA.C, Median and interquartile range (IQR) of GDF-15 concentrations for CLIA and ELISA.

Fig. 3 .
Fig. 3. Reliability of our chemiluminescence immunoassay was tested by analyzing serum GDF-15 levels from HC (healthy control), MS (metabolic syndrome), ACS (acute coronary syndrome), AF (atrial fibrillation), CHF (chronic heart failure) groups.Statistical analysis of blood serum data was conducted using Mann Whitney test.